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. 2019 Aug 13:13:374.
doi: 10.3389/fncel.2019.00374. eCollection 2019.

Lack of Neuronal Glycogen Impairs Memory Formation and Learning-Dependent Synaptic Plasticity in Mice

Jordi Duran  1   2 , Agnès Gruart  3 , Olga Varea  1 , Iliana López-Soldado  1   2 , José M Delgado-García  3 , Joan J Guinovart  1   2   4
Affiliations

Lack of Neuronal Glycogen Impairs Memory Formation and Learning-Dependent Synaptic Plasticity in Mice

Jordi Duran et al. Front Cell Neurosci. .

Abstract

Since brain glycogen is stored mainly in astrocytes, the role of this polysaccharide in neurons has been largely overlooked. To study the existence and relevance of an active neuronal glycogen metabolism in vivo, we generated a mouse model lacking glycogen synthase specifically in the Camk2a-expressing postnatal forebrain pyramidal neurons (GYS1Camk2a-KO), which include the prefrontal cortex and the CA3 and CA1 cell layers of the hippocampus. The latter are involved in memory and learning processes and participate in the hippocampal CA3-CA1 synapse, the function of which can be analyzed electrophysiologically. Long-term potentiation evoked in the hippocampal CA3-CA1 synapse was decreased in alert behaving GYS1Camk2a-KO mice. They also showed a significant deficiency in the acquisition of an instrumental learning task - a type of associative learning involving prefrontal and hippocampal circuits. Interestingly, GYS1Camk2a-KO animals did not show the greater susceptibility to hippocampal seizures and myoclonus observed in animals completely depleted of glycogen in the whole CNS. These results unequivocally demonstrate the presence of an active glycogen metabolism in neurons in vivo and reveal a key role of neuronal glycogen in the proper acquisition of new motor and cognitive abilities, and in the changes in synaptic strength underlying such acquisition.

Keywords: LTP; glycogen; learning; memory; metabolism.

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Figures

FIGURE 1
FIGURE 1
Analysis of GYS1 expression in brain regions of control and GYS1Camk2a–KO mice. (A) qPCR analysis of GYS1 gene expression. The expression of GYS1 was measured in the cortex, hippocampus and cerebellum of control (n = 4) and GYS1Camk2a–KO mice (n = 5). The data shown are mean ± SEM of 2^ddCt in relative units for each genotype analyzed. Significant differences between groups were found in the cortex (p < 0.0001) and hippocampus (p = 0.0383), but not in cerebellum. (B) Western blot analyses of GYS1 protein and quantification of the results. Protein homogenates of the same regions were analyzed by western blot with an antibody specific for GYS1 protein (n = 4 per region and genotype). Again significant differences between groups were found in the cortex (p = 0.0006) and hippocampus (p = 0.0474), but not in the cerebellum. Statistics were calculated using unpaired t-test, statistical values. *p < 0.05, ∗∗∗p < 0.001.
FIGURE 2
FIGURE 2
Electrophysiological properties of hippocampal synapses in alert behaving control and GYS1Camk2a–KO mice. (A) Experimental design. Animals were chronically implanted with stimulating electrodes in CA3 Schaffer collaterals and with a recording electrode in the ipsilateral CA1 area. An extra wire was attached to the bone as ground. DG, dentate gyrus; Sub., subiculum. Adapted from Duran et al. (2013). (B) Input/output curves of fEPSPs evoked at the CA3-CA1 synapse by paired (40 ms of inter-pulse interval) pulses of increasing intensities (0.02–0.4 mA) in control (n = 8; black circles) and GYS1Camk2a–KO (n = 8; white circles) mice. Data are represented as mean ± SEM. No significant differences [F(19,266) = 0.579; p = 0.92] were observed between groups. (C) In addition, no significant [F(5,110) = 0.753; p = 0.586] differences in paired-pulse facilitation between control (n = 12; black circles) and GYS1Camk2a–KO (n = 12; white circles) mice were detected. The data shown are mean ± SEM slopes of the 2nd fEPSP expressed as a percentage of the first of six (10, 20, 40, 100, 200, 500) inter-pulse intervals. Selected fEPSP paired traces (40 ms of inter-pulse interval) collected from representative control and GYS1Camk2a–KO mice are shown at the top right. (D) The two graphs illustrate the time course of LTP evoked in the CA3-CA1 synapse (fEPSP mean ± SEM) of control (n = 14; black circles) and GYS1Camk2a–KO (n = 14; white circles) mice following a HFS session. The HFS was presented after 15 min of baseline recordings, at the time marked by the dashed line. LTP evolution was monitored over 5 days. At the top, illustrated representative examples of fEPSPs from representative control and GYS1Camk2a–KO mice collected at the times indicated in the bottom graphs. fEPSP slopes are given as a percentage of fEPSP values collected during baseline recordings (100%). Although the two groups presented significant (p < 0.001) increases (ANOVA, two-tailed) in fEPSP slopes after HFS when compared with baseline recordings, the control group showed a larger and longer lasting LTP [F(38,988) = 2.049; p < 0.001] than GYS1Camk2a–KO mice. fEPSP slopes collected from control animals were significantly larger than those from GYS1Camk2a–KO mice at the indicated times (*p < 0.05; ∗∗p < 0.01).
FIGURE 3
FIGURE 3
Performance of control and GYS1Camk2a–KO mice in an operant conditioning task. (A) Experimental setup. Mice were trained in a Skinner box to press a lever to obtain a food pellet, with a fixed-ratio (1:1) schedule. Adapted from Duran et al. (2013). (B) Animals were trained with two programs of increasing difficulty. As illustrated in the top diagram, they were first trained to acquire a fixed-ratio (1:1) schedule until obtaining 20 pellets/20 min session on two successive days (criterion). Afterward, lever presses were rewarded only when a light bulb was switched on (see bottom diagram). (C) Time to reach the selected criterion for control (n = 13) and GYS1Camk2a–KO (n = 15) mice (t = 239.000; p = 0.017; Shapiro–Wilk t test). (D) Data collected from the first 2 days after reaching the criterion with the fixed-ratio (1:1) schedule. Illustrated data correspond to the mean ± SEM collected from control (n = 13) and GYS1Camk2a–KO (n = 15) animals. Note that although GYS1Camk2a–KO mice pressed the lever more frequently than the control group, there were no significant differences between groups (U = 58.5; p = 0.13; Mann–Whitney U statistic test). (E) Performance of control (n = 13) and GYS1Camk2a–KO (n = 14) mice during the light/dark test. Note that although both groups showed an improvement in performance across sessions [F(1,225) = 10.49; p < 0.01], control mice outperformed GYS1Camk2a–KO mice [F(9,225) = 2.82; p = 0.01]. The light/dark coefficient was calculated as follows: (number of lever presses during the light period – number of lever presses during the dark period)/total number of lever presses. For individual sessions *p = 0.05; ∗∗p = 0.01.
FIGURE 4
FIGURE 4
Performance of control and GYS1Camk2a–KO mice in a fear conditioning task. (A) Experimental design. During the conditioning test (1), animals were allowed to explore context A for 3 min, after which they were presented with a conditioning tone for 30 s (CS). The tone co-terminated with a foot shock (0.4 mA, 2 s). As illustrated, the CS-US pairing was repeated two more times. For the context test (2), the animal was located in the same box for 6 min in absence of CS or US presentations. Finally, for the cued test (3), the animal was located in context B for a control period of 3 min, followed by 3 min with a CS presentation. (B) Diagrammatic representation of contexts A (left) and B (right). (C) No significant differences between the two groups (n = 14 control and n = 15 GYS1Camk2a–KO mice) were observed for either of context (t = 0.595; 27 degrees of freedom; p = 0.557) or the cued tests (t = 0.0166; 27 degrees of freedom; p = 0.987).
FIGURE 5
FIGURE 5
Susceptibility of control and GYS1Camk2a–KO mice to kainate. (A) RLFPs recorded in the CA1 area of representative control and GYS1Camk2a–KO mice. (B) Spectral power of LFPs (60-s segments collected from n = 12 control and n = 12 GYS1Camk2a–KO mice). The inset illustrates the global boxplot of peak spectral powers [F(1,22) = 0.07; p = 0.7908]. (C) Representative examples of hippocampal seizures evoked in control and GYS1Camk2a–KO mice following the administration of 8 mg/kg i.p. of kainate. (D) Percentage of control (n = 13) and GYS1Camk2a–KO (n = 15) mice presenting spontaneous seizures at the CA1 area during the recording period (60 min). No significant differences between groups (Chi-square = 0.506 with 1 degree of freedom; p = 0.477) were observed. (E) Mean duration of kainate-evoked seizures in control and GYS1Camk2a–KO mice. No significant differences between groups were observed [F(1,14) = 0.0604; p = 0.809; ANOVA and Shapiro–Wilk test].
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